Androgenic switch in barley microspores
De Faria Maraschin, Simone
Citation
De Faria Maraschin, S. (2005, February 9). Androgenic switch in barley microspores.
Retrieved from https://hdl.handle.net/1887/606
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Corrected Publisher’s Version
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Licence agreement concerning inclusion of doctoral thesis in the
Institutional Repository of the University of Leiden
of barley m icrospore androgenesis
Biologia (2003) 58: 59-68
Abstract
In the barley (Hordeum vulgare L.) anther, tapetum and loculus wall cells undergo programmed cell death (PCD) at the time around the first pollen mitosis, at the uninucleate stage of microspore development. This is the stage where androgenesis is most efficiently induced in barley microspores. Induction of androgenesis is characterized by a switch of the normal pollen developmental pathway towards an embryogenic route via a stress pre-treatment of anthers for 4 days in mannitol solution. We were interested in studying the involvement of members of the 14-3-3 family of regulatory proteins during barley androgenesis induction. With the use of isoform-specific antibodies against the three 14-3-3 isoforms, 14-3-3A, 14-3-3B and 14-3-3C, we have studied their immunolocalization and expression level in anthers. All isoforms were localized in the microspores and in anther wall cells at the induction stage. At this period, 14-3-3A processing was found to take place in tapetum and loculus wall cells, where in situ DNA fragmentation was detected by TUNEL assay. After 4 days pre-treatment to induce androgenesis, anther wall cells degenerated and two types of morphologically distinct microspores were observed, enlarged and non-enlarged cells. At this stage, 14-3-3 isoforms were mainly localized in the microspores. 14-3-3A processing was found to be induced by stress and it could only be detected in non-enlarged cells with decreased viability after pre-treatment. Viable enlarged cells and pollen under normalin vivo development showed no visible 14-3-3A processing. The identification of 14-3-3A processing in anther wall cells and in microspores with decreased viability represents the first link between the processing of a specific 14-3-3 isoform in cells undergoing death pathway. The implications of this post-translational event in barley anthers are discussed.
Introduction
under PCD. It includes nuclear condensation of chromatin, DNA fragmentation, shrinkage of nucleus and loss of cell shape and integrity (Danon et al., 2000).
In animal cells, proteins involved in the apoptotic pathway are known to be regulated by members of the 14-3-3 protein family (Fu et al., 2000). 14-3-3s are highly conserved dimeric proteins with a subunit mass of approximately 30kD shown to be ubiquitously expressed in eukaryotes. In multicellular organisms, 14-3-3 proteins are present in many isoforms and take part of several processes within the cell. Besides their involvement in the control of apoptosis, 14-3-3s have been reported to participate in the regulation of cell cycle, intracellular signaling, transcription activation, vesicle trafficking and primary metabolism of the eukaryotic cell (van Hemert, 2001). Their regulatory functions arise from their properties to bind phosphopeptide motifs contained in their interacting proteins, which are exerted by the conserved core region of the monomers (Yaffe et al., 1997). The N-terminal dimerization domain and the hyper variable C-terminus, however, show little homology among different isoforms (Wang and Shakes, 1996). No specific functions have been so far described for the unconserved C-terminal region of 14-3-3 proteins.
In barley, three 14-3-3 isoforms have been cloned: 14-3-3A (GenBank X62388; Brandt et al., 1992), 14-3-3B (GenBank X93170) and 14-3-3C (GenBank Y14200). It has been reported that a 14-3-3A 28 kD band is formed by proteolytic cleavage of the unconserved C-terminal region of the 30 kD protein at the positions Lys 250/ Ala 255 upon the germination of barley embryos (van Zeijl et al., 2000; Testerink et al., 2001). During the onset of germination of barley embryos, massive TUNEL positive staining has been observed (unpublished data) and PCD has been described in barley scutellum and aleurone layer (Lindholm et al., 2000; Wang et al., 1996; Wang et al., 1998). It is known that several proteinases become active during barley germination which take part of the breakdown of storage proteins (Gibbons, 1980; Palmer, 1982; Briggs, 1987). Germination-related proteinases and their putative involvement in plant PCD have been recently reviewed by Beers et al. (2000). So far, no causal link has been established between PCD and the processing of 14-3-3A. The concomitant occurrence of PCD and 14-3-3A proteolytic cleavage during germination challenged us to further study this inter-relationship.
nuclear chromatin, DNA fragmentation, followed by nucleus and cell shrinkage (Papini et al., 1999; Wang et al., 1999). However, contrasting PCD in tapetum cells that is accomplished during pollen maturation, Wang et al. (1999) described massive PCD in tapetum cells in response to osmotic and starvation stresses upon androgenesis induction in barley microspores. During barley androgenesis induction, microspores are switched from their normal pollen development towards an embryogenic route by pre-treating whole anthers containing microspores at the mid-late to late uninucleate stage of development for 4 days in a mannitol solution (Hoekstra et al., 1992). The starvation and osmotic stresses imposed have been described as the triggers for the developmental switch observed during barley androgenesis (Wang et al., 2000).
We have used barley androgenesis as a model system to study 14-3-3 proteins and to investigate whether 14-3-3A processing was present in the developmental switch of microspores and in the PCD of anther tissues. To do so, isoform-specific antibodies against the three barley 14-3-3 isoforms (Testerink et al., 1999) were used to study 14-3-3 localization and expression levels in barley anthers. For the study of 14-3-3A processing, we have used two 14-3-3A antibodies, which were raised against the synthetic peptides 237-250 and 251-261. The former is able to detect both the 30 kD and the 28 kD forms of 14-3-3A, while the latter detects only the 30 kD unprocessed form of 14-3-3A due to the loss of the antibody recognition site by processing (Testerink et al., 1999; Testerink et al., 2001).
Materials and Methods
Androgenesis induction and microspore isolation
carried out in 0.37 M mannitol solution, and microspores from control anthers were isolated in 8.5 % maltose solution. In order to obtain homogeneous populations of enlarged and non-enlarged microspores after anther incubation for 4 days in 0.37 M mannitol, microspores were loaded on a 15 % (w/v) sucrose gradient in 0.37 M mannitol solution and centrifuged at 800 rpm for 10 minutes for separation of enlarged and non-enlarged cells.
Protein isolation and W estern analysis
Total protein extracts were prepared from anthers at day 0 and at day 4 of pre-treatment. Protein extracts were obtained from whole anthers gently macerated with the use of a metal pestle in protein extraction buffer at room temperature. Under such isolation conditions, microspores remained intact, which was checked under light microscope. These extracts contained mainly proteins from anther tissue. Protein extracts were prepared from purified enlarged and non-enlarged microspores after 4 days pre-treatment, binucleate pollen and microspores pre-treated for 0, 4 h, 12 h, and 1, 2, 3 and 4 days by vigorous maceration of the cells with a glass pestle in protein extraction buffer at room temperature. Protein extraction buffer consisted of 60 mM Tris pH 6.8, 10 % Glycerol, 5 % ȕ-mercaptoethanol and 2 % SDS. The extracts were heated for 10 min at 95-100ºC and centrifuged two times at 14,000 rpm for 10 minutes to collect supernatant. Soluble proteins (10 µg) were separated on 15 % (w/v) SDS-PAGE and blotted onto nitrocellulose membranes or stained with coomassie brilliant blue 0.1 % (w/v) in 40 % (v/v) methanol and 10 % (v/v) acetic acid. Blots were incubated overnight at 4ºC with isoform-specific 14-3-3 antibodies (1:20,000). Two 14-3-3A antibodies, raised against synthetic peptides 237-250 and 251-261 were used (Testerink et al., 1999; Testerink et al., 2001). Anti-14-3-3B and anti-14-3-3C antibodies were raised against peptides 248-262 and 251-262, respectively (Testerink et al., 1999). Bands were visualized by goat anti-rabbit horseradish peroxidase conjugate (Promega), followed by enhanced chemoluminescent detection (ECL) (Amersham).
Immunolocalization studies
Anthers containing microspores at the mid-late to late uninucleate stage and 4 days pre-treated anthers were fixed in 4 % paraformaldehyde in 10 mM NaH2PO4, 120 mM NaCl,
polymerised in Beem capsules under UV light for 48 h at -20ºC. Sections (5 µm) were attached to 2 % 3-aminopropiltriethoxy silane (Sigma) coated slides. After removal of the resin by acetone, proteins were denatured 20 minutes in 0.4 % (w/v) SDS, 3 mM ȕ-mercaptoethanol, 12 mM Tris pH 6.8 and blocked 30 minutes in 1 % (w/v) bovine serum albumine (BSA) in PBS buffer, pH 7.4. Primary antibody incubation was carried out overnight at 4ºC in 0.01 % acetylated BSA (Aurion) in PBS buffer, pH 7.4 (anti-14-3-3A diluted 1:1,000; anti-14-3-3B diluted 1:5,000 and anti-14-3-3C diluted 1:5,000). Sections were developed with alkaline-phosphatase-conjugated goat anti-rabbit antibody (Promega). The signal was visualized by incubating sections in nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) substrate (Promega). Control experiments were performed omitting the first antibody. Slides were mounted in a mixture of 166 g/L polyvinylalcohol and 30 % (v/v) glycerol in PBS buffer, pH 7.4 and analyzed by light microscopy.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)
Anthers at the mid-late to late uninucleate stage and after 4 days incubation in mannitol solution were fixed in 2 % glutaraldehyde in 10 mM NaH2PO4, 120 mM NaCl, 2.7
mM KCl, pH 7.4 (phosphate-buffered saline, PBS) overnight at room temperature. After dehydration at room temperature in a graded series of 70 %, 90 %, 96 % and 100 % (v/v) ethanol, samples were embedded in Historesin. Sections (2 µm) were attached to Biobond (Biocell) coated slides. TUNEL staining was done using an in situ cell death detection kit (Boehringer) and analysed by fluorescence microscopy (Wang et al., 1999). Following TUNEL reaction, nuclear staining was performed by incubating sections in 4’ -6-diamidino-2-phenylindole (DAPI) 0.02 mg/ml in water for 5 minutes at room temperature.
FDA staining
Isolated microspores after androgenesis induction were stained for viability with fluorescein diacetate (FDA) 0.04 µg/ml in acetone for 10 minutes at room temperature and analysed by fluorescence microscopy.
Results
14-3-3A processing in pre-treated microspores
pre-treatment. Upon androgenesis induction, 14-3-3A, 14-3-3B and 14-3-3C were present at higher levels in the isolated microspores (Fig. 1a-c). Moreover, in extracts of microspores at the uninucleate stage of development, mainly a 30 kD band of 14-3-3A was detected (Fig. 1a; day 0), whereas microspores after pre-treatment displayed both forms of 14-3-3A, a band at 30 kD and a band at 28 kD (Fig. 1a; day 4). This suggested that processing of 14-3-3A in the microspores was taking place during stress pre-treatment. Therefore, we monitored 14-3-3A protein expression during the course of pre-treatment. The 28 kD form of 14-3-3A could already be detected in microspores isolated from anthers that have been pre-treated for 4 hours in mannitol solution (Fig. 2). Interestingly, the 28 kD processed form of 14-3-3A seemed not to be a feature of normal pollen development, as mainly a 30 kD band was detected in extracts of binucleate pollen (Fig. 2; lane B).
Figure 1. Western blot analysis of 14-3-3 expression in isolated microspores upon androgenesis induction. Microspores were isolated from anthers at day 0 and day 4 of pre-treatment as indicated above the lanes. 10 µg/lane of total protein extracts were separated by SDS-PAGE. Proteins were blotted onto nitrocellulose membranes and incubated with the isoform-specific antibodies. Signal was detected by enhanced chemoluminescent methods. Protein loading was verified by coomassie staining. Blots were incubated with anti 14-3-3A raised against synthetic peptide 237-250 (a), anti-14-3-3B (b) and anti-14-3-3C (c). One representative blot from 4 independent experiments is shown for each 14-3-3 isoform.
Upon pre-treatment, 60 % of the microspores appear as enlarged, vacuolated cells. Microspores displaying this morphology have been often reported to have acquired embryogenic potential after pre-treatment, while the remaining microspore population represents non-enlarged cells with no ability to divide during further microspore culture (Hoekstra et al., 1993). We were interested in further investigating whether the 14-3-3A processing was a feature of these two microspore populations. To do so, we separated microspores that have been pre-treated for 4 days by a sucrose gradient in order to obtain isolated fractions of enlarged and non-enlarged cells. In the population of enlarged microspores, 14-3-3A was present as its 30 kD form, while in non-enlarged microspores the 28 kD processed form of 14-3-3A was mainly detected (Fig. 3a). Processing of 14-3-3A by proteolytic cleavage of the unconserved C-terminus of the protein (van Zeijl et al., 2000; Testerink et al., 2001) was confirmed by the use of the 14-3-3A antibody that recognizes the amino acids 251-262. The antibody detected only the 30 kD 14-3-3 A form in enlarged and non-enlarged microspores (Fig. 3b), thus confirming the loss of the unconserved C-terminus of the 14-3-3A protein. FDA staining of the separated enlarged and non-enlarged microspore populations revealed the presence of FDA positive cells only in the enlarged microspore population, indicating that they were alive (Fig. 4a,b). Non-enlarged microspores were negatively stained for FDA and represented probably dying cells after pre-treatment (Fig. 4c,d).
In microspores after pre-treatment, the processing of 14-3-3A was associated to decreased viability of the non-enlarged cells (Fig. 3, 4). The question raised is whether cell death pathway of microspores upon pre-treatment shows features of PCD. We have used the TUNEL reaction (Gavrieli et al., 1992), which labels the 3’ ends of DNA strand breaks, to assess in situ DNA fragmentation in the anthers upon the induction of androgenesis (Fig. 5).
Figure 4. Microspore viability in enlarged and non-enlarged microspores after 4 days of mannitol pre-treatment assessed by FDA staining. The two populations were separated by a 15% sucrose gradient. (a) Enlarged microspores observed by light micrsocopy and (b) by fluorescence microscopy stained for FDA. (c) Non-enlarged microspores observed by light microscopy and (d) by fluorescence microscopy stained for FDA. Bars: 100 Pm.
In anthers containing microspores at the mid-late to late uninucleate stage, TUNEL positive nuclei were observed mainly in anther tapetum, loculus and epidermis (Fig. 5a). Upon stress pre-treatment, we observed occurrence of TUNEL positive nuclei in some microspores, in the epidermal tissue and in the outermost layer of loculus wall cells, whereas anther tapetum and the innermost layer of loculus wall cells had degenerated (Fig. 5b).
14-3-3 immunolocalization and detection of 14-3-3A processing in anthers
Upon osmotic and starvation stresses, anther wall cells and microspores underwent several morphological changes (Fig. 6). After pre-treatment, the anther tapetum and the innermost layer of loculus wall cells degenerated, while some of the microspores inside the anther appeared as enlarged, vacuolated cells (Fig. 6e-h). We studied the 14-3-3 immunolocalization in freshly isolated barley anthers containing microspores at the verge of mitosis, and investigated their temporal and spatial expression in anthers that have been pre-treated for the induction of androgenesis.
The immunolocalization patterns of the three barley 3 isoforms, 3A, 14-3-3B and 14-3-3C are shown in Figure 6. In freshly isolated anthers at the stage of androgenesis induction, 14-3-3 isoforms were present both in the microspores and in the anther wall cells. In the microspores, 14-3-3 signals were mainly detected in the cytoplasm of the cells. In the anther wall, all three isoforms were expressed in tapetum and in the loculus wall cells (Fig. 6a-c), however 14-3-3A signal was stronger in the tapetum layer when compared to that of 14-3-3B and 14-3-3C. After anther pre-treatment for the induction of androgenesis, 14-3-3 immunolocalization was restricted to the microspores. No signal was detected in the outermost layer of loculus wall cells, while the tapetum and the innermost layer of loculus wall cells had degenerated (Fig 6e-g). In pre-treated microspores, 14-3-3 signals were stronger than in untreated microspores, thus confirming the higher protein levels of the 14-3-3 isoforms observed by western blot analysis (Fig. 1). No signal was detected in the control sections incubated only with the secondary antibody (Fig. 6d,h).
Next, we investigated 14-3-3 protein expression levels in anther tissues before and after stress pre-treatment. In extracts of anthers developed to the stage of uninucleate microspores, 14-3-3A was present as a double band, at 30 kD and 28 kD respectively (Fig. 7a; day 0). 14-3-3B and 14-3-3C were present as bands of approximately 31 kD (Fig. 7b,c; day 0). The presence of a 28 kD form of 14-3-3A indicates that processing is a feature of anthers that have developed to the stage of uninucleate microspores.
Figure 6. Immunolocalization of 14-3-3 isoforms in anthers analysed by light microscopy. Cross-sections of anthers containing microspores at the mid-late to late uninucleate stage (a) (b) (c) (d) and after 4 days of pre-treatment in mannitol solution (e) (f) (g) (h). (a/e) show immunolocalization of 3A, (b/f) 14-3-3B and (c/g) 14-3-3C. (d/h) were incubated only with secondary antibody. e epidermis, il innermost layer of loculus wall cells, ol outermost layer of loculus wall cells, m microspores, t tapetum. Remains of tapetum and innermost layer of loculus wall cells are indicated by (*). 14-3-3 immunolocalization was studied in at least 5 anthers in 3 independent experiments. Representative examples are shown for each 14-3-3 isoform. Bars: 20 Pm.
Discussion
14-3-3A processing during PCD in tapetum upon normal pollen development
In germinating barley embryos, the 14-3-3A isoform is processed into a lower molecular weight form of 28 kD by proteolytic cleavage of the unconserved C-terminus of the protein (van Zeijl et al., 2000; Testerink et al., 2001). TUNEL positive staining has been observed in the germination process of barley (unpublished data). However, no link between processing of 14-3-3A and the death of specific cell types could be established in the above studies. We have demonstrated that both the 30 kD and the 28 kD forms of 14-3-3A were immunolocalized in tapetum and loculus wall cells in developing anthers where the microspores were at the mid-late to late uninucleate stage of pollen development (Fig. 6a, 7A). PCD has been demonstrated to take place in the anther tapetum and loculus wall cells by Wang et al. (1999) and we have shown that these are the cell layers to display DNA fragmentation in TUNEL assay (Fig 5a). Our results consist in the first report showing 14-3-3A processing in cell types dying in a programmed way. It is possible that 14-3-14-3-3A processing is linked to a PCD-related proteolytic cleavage.
Several proteinases have been identified during PCD of anther wall cells. In Solanum melongena, the expression of a cysteine proteinase mRNA has been correlated to the death of endothecium upon anther dehiscence (Xu and Chye, 1999), whereas accumulation of the transcript encoding a thiol endopeptidase has been described in dehiscent tobacco anthers (Koltunow et al., 1990). The LIM9 serine proteinase, whose putative function is to take part in PCD, is synthesized as a preprotein in the young tapetum and is secreted in the anther loculus, where it is also expressed in the microspores (Taylor et al., 1997). The expression of PCD-related proteinases upon the death of anther wall layers reinforce the idea that 14-3-3A processing is related to a cell death pathway. However, the biological function of the 14-3-3A processing is not yet clear. Both 14-3-3 A 30 kD and 28 kD forms were found to bind to the plasma membrane H+- ATPase in in vitro gel overlay assays (Testerink et al., 2001), suggesting that the 28 kD is still functional. van Zeijl et al. (2000) observed the 28 kD form of 14-3-3A to accumulate in cytosolic and microsomal fractions of germinating barley embryos, an indication that 14-3-3A processing might affect its subcellular localization. The characterization of the 3A proteinase will help clarifying whether 14-3-3A processing is accomplished by a PCD-related proteinase and it will shed light on its biological function.
14-3-3A processing during the developmental switch of barley microspores
allowed to develop for further 4 days in the mother plant (Fig. 1a; 2). This suggests that 14-3-3A processing may not be important for in vivo development and it is probably not required for the first pollen mitosis. However, one cannot exclude 14-3-3A processing to take place in the microspores beyond our detection level.
Acknowledgments
We are grateful to Sandra van Bergen for technical assistance and to Bert van Duijn for critical reading of the manuscript.
References
Alché JD, Castro, AJ, Solymoss M, Timar I, Barnabas B, Rodríguez-García MI (2000) Cellular approach to the study of androgenesis in maize anthers: immunocytochemical evidence of the involvement of the ubiquitin degradative pathway in androgenesis induction. J Plant Physiol 155: 146-155 Beers EP, Bonnie J, Zhao W, Zhao C (2000) Plant proteolytic enzymes: possible roles during
programmed cell death. Plant Mol Biol 44: 399-415
Brandt J, Thordal-Christensen H, Vad K, Gregersen PL, Collinge DB (1992) A pathogen-induced gene of barley encodes a protein showing high similarity to a protein kinase regulator. Plant J 2: 815-820 Briggs DE (1987) Endosperm breakdown and its regulation in barley germination. In: Schweigert S (ed),
Food and Science Technology, Academic Press, London, pp. 441-532
Callard D, Axelos M, Mazzolini L (1996) Novel molecular markers for late phases of the growth cycle of Arabidopsis thaliana cell-suspension cultures are expressed during organ senescence. Plant Physiol 112: 705-715
Cordewener J, Bergervoet J, Liu CM (2000) Changes in protein synthesis and phosphorylation during microspore embryogenesis in Brassica napus. J Plant Physiol 156: 156-163
Danon A, Delorme V, Mailhac N, Gallois P (2000) Plant programmed cell death: A common way to die. Plant Physiol Biochem 38: 647-655
Fu H, Subramanian RR, Masters SC (2000) 14-3-3 proteins: structure, function and regulation. Annu Rev Pharmacol Toxicol 40: 617-647
Gibbons GC (1980) On the sequential determination of Į-amylase transport and cell breakdown in germinating seeds of Hordeum vulgare. Carlsberg Research Communication 45: 177-184 Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific
labelling of DNA fragmentation. J Cell Biol 119: 493-501 Green DR, Beere HM (2001) Mostly dead. Nature 412: 133-135
Häcker G (2000) The morphology of apoptosis. Cell Tissue Res 301: 5-17
Hoekstra S, van Zijderveld MH, Heidekamp F, van der Mark F (1993) Microspore culture of Hordeum vulgare L.: the influence of density and osmolarity. Plant Cell Rep 12: 661-665
Hoekstra S, van Zijderveld MH, Louwerse JD, Heidekamp F, van der Mark F (1992) Anther and microspore culture of Hordeum vulgare L. cv. Igri. Plant Sci 86: 89-96
Katsuhara (1997) Apoptosis-like cell death in barley roots under salt stress. Plant Cell Physiol 38: 1091-1093
Koltunow AM, Truettner J, Cox KH, Wallroth M, Goldberg RB (1990) Different temporal and spatial expression patterns occur during anther development. Plant Cell 2: 1201-1224
Kyo M, Harada H (1990a) Phosphorylation of proteins associated with embryogenic dedifferentiation of immature pollen grains of Nicotiana rustica. J Plant Physiol 136: 716-722
Kyo M, Ohkawa T (1991) Investigation of subcellular localization of several phosphoproteins in embryogenic pollen grains of tobacco. J Plant Physiol 137: 525-529
Lindholm P, Kuittinen T, Sorri O, Guo D, Merits A, Törmäkangas K, Runeberg-Roos P (2000) Glycosylation of phytepsin and expression of dad1, dad2 and ost1 during onset of cell death in germinating barley scutella. Mech Develop 93: 169-173
McCormick S (1993) Male Gametophyte Development. Plant Cell 5: 1265-1275
Palmer GH (1982) A reassessment of the pattern of endosperm hydrolysis (modification) in germinated barley. Journal of the Institute of Brewing 88: 145-153
Papini A, Mosti S, Brighigna L (1999) Programmed cell death events during tapetum development of angiosperms. Protoplasma 207: 213-221
Raff M (1998) Cell suicide for beginners. Nature 396: 119-122
Taylor AA, Horsch A, Rzepczyk A, Hasenkampf CA, Riggs CD (1997) Maturation and secretion of a serine proteinase is associated with events of late microsporogenesis. Plant J 12: 1261-1271 Testerink C, van Zeijl MJ, Drumm K, Palmgren MG, Kijne JW, Wang M (2001) Post-translational
modification of barley 14-3-3A is isoform-specific and involves the removal of the hypervariable C-terminus. Plant Mol Biol 50: 535-542
Testerink C, van der Meulen RM, Oppedijk BJ, de Boer AH, Heimovaara-Dijkstra S, Kijne JW, Wang M (1999) Differences in spatial expression between 14-3-3 isoforms in germinating barley embryos. Plant Physiol 121: 81-87
van Hemert M (2001) Characterization of the Saccharomyces cerevisiae Fin1 protein: a binding partner of the 14-3-3 proteins. PhD thesis. Leiden University, Leiden, 143 pp
van Zeijl MJ, Testerink C, Kijne JW, Wang M (2000) Subcellular differences in post-translational modification of barley 14-3-3 proteins. FEBS Lett 473: 292-296
Wang M, Hoekstra S, van Bergen S, Lamers GEM, Oppedijk BJ, van der Heijden MW, de Priester W, Schilperoort RA (1999) Apoptosis in developing anthers and the role of ABA in this process during androgenesis in Hordeum vulgare L. Plant Mol Biol 39: 489-501
Wang M, Oppedijk BJ, Caspers MPM, Lamers GEM, Boot MJ, Geerlings DNG, Bakhuizen B, Meijer AH, van Duijn B (1998) Spatial and temporal regulation of DNA fragmentation in the aleurone of germinating barley. J Exp Bot 325: 1293-1301
Wang M, Oppedijk BJ, Lu X, van Duijn B, Schilperoort RA (1996) Apoptosis in barley aleurone during germination and its inhibition by abscisic acid. Plant Mol Biol 32: 1125-1134
Wang M, van Bergen S, van Duijn B (2000) Insights into a key developmental switch and its importance for efficient plant breeding. Plant Physiol 124: 523-530
Wang W, Shakes DC (1996) Molecular evolution of the 14-3-3 protein family. J Mol Evol 43: 384-398 Wu H, Cheung AY (2000) Programmed cell death in plant reproduction. Plant Mol Biol 44: 267-281 Xu FX, Chye ML (1999) Expression of cysteine proteinase during developmental events associated with
programmed cell death in brinjal. Plant J 17: 321-327
